Recent studies underscore the lackluster performance of cancer diagnostics, including measurement of prostate specific antigen (PSA) in prostate cancer. PSA levels measured routinely as a minimally invasive blood test may inaccurately reflect disease. To advance our capacity to identify & treat prostate cancer, new indicators of disease state, therapeutic response and outlook are needed. Prostate circulating tumor cells (CTCs) offer potentially powerful new biomarkers for understanding cancer. The impetus for single-CTC analysis stems from the observed cellular heterogeneity in solid tumor microenvironments. CTCs are believed to initiate metastasis, accounting for approximately 90% of deaths from solid tumors. Previous studies link CTC enumeration to patient outcome. CTC analysis may provide insight into therapies. While molecular analyses of single-cells has advanced tremendously over the last decade, revealing genetic diversity in tumor cell subpopulations, a complete understanding of cancer progression and variability requires tools to assess protein-mediated signaling directly. A major limitation on querying CTCs for protein- level information stems from the staggeringly low number of CTCs found in blood (i.e., 1 CTC per billion normal blood cells). Out of necessity, protein assays are performed on CTC populations, ignoring the inherent variability between cells. Yet, we know that cell-to-cell variability may hold information crucial to understanding the initiation and final stages of metastasis, as well as in identifying promising drug targets and candidates for stemming metastasis. In conjunction with our clinical collaborators at Stanford, we will significantly advance and expand on our recently introduced single-cell microfluidic Western blot array to quantify protein expression in single CTCs collected via MagSweeper from prostate cancer patients. Our work will enable first-in-kind Western blotting of single CTCs. To do this, we will: (1) introduce a single-cell scWestern array optimized for low starting numbers of CTCs (10-100 cells), in tandem with introducing high sensitivity readouts, (2) apply single CTC Western blotting to analyses of differential expression of targeted proteins in a suite of cell types with increasing degree of hypothesized metastatic potential, and (3) introduce fluid flow control to the single-CTC Western array for in situ cell surface antigen staining to allow correlation with intracellular protein-mediated signaling. Taken together, our work will advance cancer research by allowing for inclusion of CTC-level protein data. Looking forward, the new knowledge gained as a result of our IMAT success would notably advance clinical practice by enabling study of promising CTC biomarkers of sensitivity/acquired resistance to novel cancer therapies and in identifying potential therapeutic targets to halt cancer metastasis. Protein-level characterization of CTCs is important to realizing truly personalized medicine.